Links

Images

Classifications

C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom

C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring

C07D209/04—Indoles; Hydrogenated indoles

C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring

C07D209/32—Oxygen atoms

C07D209/34—Oxygen atoms in position 2

Abstract

The present invention relates to novel geometrically restricted 2-indolinones and physiologically acceptable salts thereof which modulate the activity of protein kinases and therefore are expected to be useful in the prevention and treatment of protein kinase related cellular disorders such as cancer.

Description

This application is a division of application Ser. No. 09/385,974 filed Aug. 30, 1999 now U.S. Pat. No. 6,525,072.

RELATED APPLICATIONS

This application is related to and claims priority from United States Provisional Patent Application serial No. 60/098,660 filed Aug. 31, 1998 which is incorporated by reference as if fully set forth herein.

INTRODUCTION

This invention relates generally to organic chemistry, biochemistry, pharmacology and medicine. More particularly, it relates to geometrically restricted 2-indolinone derivatives and their physiologically acceptable salts and prodrugs which modulate the activity of protein kinases (“PKs”) and, therefore, are expected to exhibit a salutary effect against disorders related to abnormal PK activity.

BACKGROUND OF THE INVENTION

The following is offered as background information only and is not admitted to be prior art to the present invention.

PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple activity are staggering; cell growth, differentiation and proliferation, i.e., virtually all aspects of cell life in one way or another depend on PK activity. Furthermore, abnormal PK activity has been related to a host of disorders, ranging from relatively non-life threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).

The PKs can be conveniently broken down into two classes, the protein tyrosine kinases (PTKS) and the serine-threonine kinases (STKs).

One of the prime aspects of PTK activity is their involvement with growth factor receptors. Growth factor receptors are cell-surface proteins. When bound by a growth factor ligand, growth factor receptors are converted to an active form which interacts with proteins on the inner surface of a cell membrane. This leads to phosphorylation on tyrosine residues of the receptor and other proteins and to the formation inside the cell of complexes with a variety of cytoplasmic signaling molecules that, in turn, effect numerous cellular responses such as cell division (proliferation), cell differentiation, cell growth, expression of metabolic effects to the extracellular microenvironment, etc. For a more complete discussion, see Schlessinger and Ullrich, Neuron, 9:303-391 (1992) which is incorporated by reference, including any drawings, as if fully set forth herein.

Growth factor receptors with PTK activity are known as receptor tyrosine kinases (“RTKs”). They comprise a large family of transmembrane receptors with diverse biological activity. At present, at least nineteen (19) distinct subfamilies of RTKs have been identified. An example of these is the subfamily designated the “HER” RTKs, which include EGFR (epithelial growth factor receptor), HER2, HER3 and HER4. These RTKs consist of an extracellular glycosylated ligand binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-like growth factor I receptor (IGF-1R) and insulin receptor related receptor (IRR). IR and IGF-LR interact with insulin, IGF-I and IGF-II to form a heterotetramer of two entirely extracellular glycosylated a subunits and two β subunits which cross the cell membrane and which contain the tyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growth factor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobin-like loops and an intracellular domain wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences.

Another group which, because of its similarity to the PDGFR subfamily, is sometimes subsumed into the later group is the fetus liver kinase (“flk”) receptor subfamily. This group is believed to be made up of kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1, VEGF-R2), flk-1R, flk-4 and fms-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family is the fibroblast growth factor (“FGF”) receptor subgroup. This group consists of four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well defined, it appears that the receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobin-like loops and an intracellular domain in which the tyrosine kinase sequence is interrupted by regions of unrelated amino acid sequences.

Still another member of the tyrosine kinase growth factor receptor family is the vascular endothelial growth factor (“VEGF”) receptor subgroup. VEGF is a dimeric glycoprotein similar to PDGF but has different biological functions and target cell specificity in vivo. In particular, VEGF is presently thought to play an essential role is vasculogenesis and angiogenesis.

A more complete listing of the known RTK subfamilies is described in Plowman et al., DN&P, 7(6):334-339 (1994) which is incorporated by reference, including any drawings, as if fully set forth herein.

In addition to the RTKS, there also exists a family of entirely intracellular PTKs called “non-receptor tyrosine kinases” or “cellular tyrosine kinases.” This latter designation, abbreviated “CTK,” will be used herein. CTKs do not contain extracellular and transmembrane domains. At present, over 24 CTKS in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Src subfamily appear so far to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detailed discussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which is incorporated by reference, including any drawings, as if fully set forth herein.

The serine/threonine kinases, STKs, like the CTKs, are predominantly intracellular although there are a few receptor kinases of the STK type. STKs are the most common of the cytosolic kinases; i.e., kinases that perform their function in that part of the cytoplasm other than the cytoplasmic organelles and cytoskelton. The cytosol is the region within the cell where much of the cell's intermediary metabolic and biosynthetic activity occurs; e.g., it is in the cytosol that proteins are synthesized on ribosomes.

RTKs, CTKs and STKs have all been implicated in a host of pathogenic conditions including, significantly, cancer. Other pathogenic conditions which have been associated with PTKs include, without limitation, psoriasis, hepatic cirrhosis, diabetes, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, immunological disorders such as autoimmune disease, cardiovascular disease such as atherosclerosis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explain the excessive cellular proliferation that drives tumor development relate to functions known to be PK regulated. That is, it has been suggested that malignant cell growth results from a breakdown in the mechanisms that control cell division and/or differentiation. It has been shown that the protein products of a number of proto-oncogenes are involved in the signal transduction pathways that regulate cell growth and differentiation. These protein products of proto-oncogenes include the extracellular growth factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.

Our own efforts to identify small organic molecules which modulate PK activity and which, therefore, would be expected to be useful in the treatment and prevention of disorders driven by abnormal PK activity, has led us to the discovery of a family of novel geometrically restricted 2-indolinone derivatives which exhibit PK modulating ability and which are the subject of this invention.

Thus, the present invention relates generally to novel geometrically restricted 2-indolinone derivatives and their prodrugs and physiologically acceptable salts which modulate the activity of receptor tyrosine kinases (RTKs), non-receptor protein tyrosine kinases (CTKs) and serine/threonine protein kinases (STKs). In addition, the present invention relates to -the preparation and use of pharmaceutical compositions of the disclosed compounds and their physiologically acceptable salts and prodrugs in the treatment or prevention of PK driven disorders such as, by way of example and not limitation, cancer, diabetes, hepatic cirrhosis, cardiovasacular disease such as atherosclerosis, angiogenesis, immunological disorders such as autoimmune disease and renal disease.

The terms “2-indolinone,” “indolin-2-one,” “2-oxindole” and “oxindole” are used interchangably herein; all refer to a chemical compound having the general structure:

As used herein, the above terms are deemed to include sulfur derivative; i.e., when Z=sulfur.

“Geometrically restricted” refers to the chemical structure about a double bond wherein groups attached to the double bond are set in their spatial relationship to one another by the very nature of the double bond. That is, atoms attached to a double bond must be coplanar; i.e., in the same plane as the atoms of the double bond itself. This is best demonstrated insofar as the compounds of this invention are concerned by looking at the generic structures shown in Formulas 1 and 2:

Formula 1 represents a backbone structure of a compound of this invention. It is understood that 1 is presented by way of example only and not limitation; backbone structures other than that shown in 1 are within the scope and spirit of this invention. The point to be gleaned from 1 is the relationship of the atoms attached to the double bond at the 3-position of the indolinone. In 1, ring system a and ring system b are linked to the double bond through a ring carbon. Since the atoms to either side of the linking carbon atom must be coplanar due to the double bond, and since the rings themselves are internally co-planar, the entire molecule, ring systems a and b and the double bond, are co-planar. This is in contrast to Formula 2, wherein a single bond connects ring system a′ to the double bond. Ring system a′ is therefore free to rotate about the single bond permitting the ring systems a′ and b′ to be non-co-planar, potentially even being perpendicular to one another.

A “pharmacological composition” refers to a mixture of one or more of the compounds described herein, or physiologically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and/or excipients. The purpose of a pharmacological composition is to facilitate administration of a compound to an organism.

As used herein, a “physiologically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

An “excipient” refers to an inert substance added to a pharmacological composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

A “prodrug” refers to an agent which is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. An example, without limitation, of a prodrug would be a compound of the present invention which is administered as an ester (the “prodrug”) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial.

A further example of a prodrug might be a short polypeptide, for example, without limitation, a 2-10 amino acid polypeptide, bonded through a terminal amino group to a carboxy group of a compound of this invention wherein the polypeptide is hydrolyzed or metabolized in vivo to release the active molecule.

1. THE COMPOUNDS

General Structural Features.

In one aspect, the present invention relates to a geometrically restricted 2-indolinone compound having chemical structure I, II or III:

The scope of this invention includes physiologically acceptable salts and prodrugs of the compound claimed herein.

Z is selected from the group consisting of oxygen and sulfur; r is 1, 2, 3, 4, 5, or 6; and,

n is 0 or 1.

The term “endo” refers to a double bond contained within a ring structure; for example, the double bond in the following structure is an “endo” double bond:

As used herein, the term “alkyl” refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more individually selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, cyanato, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, amino and —NR13R14, R13 and R14 being as defined above.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and, cycloheptatriene. A cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more individually selected from alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino and —NR13R14, with R13 and R14 being as defined above.

An “alkenyl” group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond.

An “alkynyl” group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, trihalo-methanesulfonamido, amino and —NR13R 14, R13 and R14 being as defined above.

As used herein, a “heteroaryl” group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole. The heteroaryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, carboxy, sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and —NR13R14, R13 and R14 being defined above.

A “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic ring may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably one or more selected from alkyl, cycloaklyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thio-carbamyl, sulfinyl, sulfonyl, C-amido, N-amido, amino and —NR13R14, with R13 and R14 being as defined above.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group, as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group, as defined herein.

A “mercapto” group refers to an —SH group.

A “alkylthio” group refers to both an S-alkyl and an —S-cycloalkyl group, as defined herein.

A “arylthio” group refers to both an —S-aryl and an —S-heteroaryl group, as defined herein.

A “carbonyl” group refers to a —C(═O)R″ group, where R″ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein.

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “cycloketone” refer to a cycloalkyl group in which one of the carbon atoms which form the ring has a “═O” bonded to it; i.e. one of the ring carbon atoms is a —C(═O)-group.

A “thiocarbonyl” group refers to a —C(═S)R″ group, with R″ as defined herein.

An “O-carboxy” group refers to a R″C(═O)O-group, with R″ as defined herein.

A “C-carboxy” group refers to a —C(═O)OR″ groups with R″ as defined herein.

As used herein, an “ester” is a C-carboxy group, as defined herein, wherein R″ is any of the listed groups other than hydrogen.

A “C-carboxy salt” refers to a —C(═O)O−M+ group wherein M+ is selected from the group consisting of lithium, sodium, magnesium, calcium, potassium, barium, iron, zinc and quaternary ammonium.

An “acetyl” group refers to a —C(═O)CH3 group.

A “carboxyalkyl” group refers to —(CH2)rC(═O)OR″ wherein r is 1-6 and R″ is as defined above.

A “carboxyalkyl salt” refers to a —(CH2)rC(═O)O−M+ wherein M+ is selected from the group consisting of lithium, sodium, potassium, calcium, magnesium, barium, iron, zinc and quaternary ammonium.

A “carboxylic acid” group refers to a C-carboxy group in which R″ is hydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “trihalomethyl” group refers to a —CX3 group wherein X is a halo group as defined herein.

A “trihalomethanesulfonyl” group refers to a X3CS(═O)2-group with X as defined above.

A “cyano” group refers to a —C═—N group.

A “cyanato” group refers to a —CNO group.

An “isocyanato” group refers to a —NCO group.

A “thiocyanato” group refers to a —CNS group.

An “isothiocyanato” group refers to a —NCS group.

A “sulfinyl” group refers to a —S(═O)R″ group, with R″ as defined herein.

A “sulfonyl” group refers to a —S(═O)2R″ group, with R″ as defined herein.

A “sulfonamido” group refers to a —S(═O)2NR13R14, with R″ and R14 as defined herein.

A “trihalomethanesulfonamido” group refers to a X3CS(═O)2NR13-group with X and R13 as defined herein.

An “O-carbamyl” group refers to a —OC(═O)NR13R14 group with R13 and R14 as defined herein.

An “N-carbamyl” group refers to a R14OC(═O)NR13— group, with R13 and R14 as defined herein.

An “O-thiocarbamyl” group refers to a —OC(═S)NR13R14 group with R13 and R14 as defined herein.

An “N-thiocarbamyl” group refers to a R14OC(═S)NR13— group, with R13 and R14 as defined herein.

An “amino” group refers to an —NR13R14 group, with R13 and R14 both being hydrogen.

A “C-amido” group refers to a —C(═O)NR13R14 group with R13 and R14 as defined herein. An “N-amido” group refers to a R13C(═O)NR14— group with R13 and R14 as defined herein.

A “nitro” group refers to a —NO2 group.

A “quaternary ammonium” group refers to a —+NR13R14R15 group wherein R13R14 and R15 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl.

A “methylenedioxy” group refers to a —OCH2O— group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.

An “ethylenedioxy” group refers to a —OCH2CH2O— group wherein the oxygen atoms are bonded to adjacent ring carbon atoms.

Another aspect of this invention is a combinatorial library of at least 10 compounds formed by reacting an oxindole having the general chemical structure:

By “reacting” is meant placing one of the above oxindoles and one of the above cycloketones in a chemical environment wherein they will interact with on another to form a covalent bond between them. In the present case, the covalent bond will ultimately be a double bond from the carbon atom at the 3-position of the oxindole to the keto carbon atom of the cycloketone.

A “combinatorial library” refers to all the compounds formed by the reaction of each compound in one dimension of a multi-dimensional array with a compound in each of the other dimensions of the multi-dimensional array. As used herein, the multi-dimensional array is two-dimensional, one dimension being all the oxindoles of this invention, the other dimension being all the cycloketones of this invention. Each oxindole may be reacted with each of the cycloketones to form a 2-indolinone. All 2-indolinone compounds formed in this manner are within the scope of this invention. Also within the scope of this invention are smaller combinatorial libraries formed by the reaction of some of the oxindoles of this invention with all of the cycloketones of this invention or all of the oxindoles with some of the cycloketones or some of the oxindoles with some of the cycloketones.

It is another aspect of this invention that a combinatorial library may be used to screen compounds of this invention for a desired activity.

By a “desired activity” is meant the ability to modulate the catalytic activity of a selected protein kinase.

By “screening” is meant to contact an entire combinatorial library of compounds or any portion thereof with one or more target protein kinases and then observe the effect of the compounds on the catalytic activity of the protein kinase.

Yet another aspect of this invention is a compound which modulates protein kinase activity, in particular RTK, CTK or STK kinase catalytic activity.

Preferred Structural Features.

A presently preferred embodiment of this invention is a compound in which:

n is 1;

A, B, D and E are carbon;

R1 is hydrogen; and,

Z is oxygen.

A further presently preferred embodiment of this invention is one in which:

n is 1;

A, B, D and E are carbon;

R1 is hydrogen;

Z is oxygen; and,

F, G, J and K are carbon.

Still another presently preferred embodiment of this invention is a compound in which:

n is 0;

A, B, D and E are carbon; and,

Z is oxygen.

A compound in which:

n is 0;

A, B, D and E are carbon;

Z is oxygen;

F is nitrogen;

R9 is hydrogen; and,

G and K are carbon

is yet another presently preferred embodiment of this invention.

A presently preferred embodiment of this invention would also be a compound in which:

n is 0;

A, B, D and E are carbon;

Z is oxygen;

K is nitrogen;

R6 is hydrogen; and,

F and G are carbon.

A further presently preferred embodiment of this invention is a compound in which one or two of F, G, J or K are independently nitrogen.

It is likewise a presently preferred embodiment of this invention that

n is 1;

A, B, D and E are carbon;

R1 is hydrogen;

Z is oxygen;

R13 is hydrogen; and,

R14 is unsubstituted lower alkyl.

It is still another presently preferred embodiment of this invention that:

lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted lower alkoxy;

lower alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″, unsubstituted aryl or —NR13R14;

trihalomethyl;

unsubstituted alkenyl;

unsubstituted alkynyl;

unsubstituted aryl;

aryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl or lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted heteroalicyclic;

heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, —C(═O)H, —C(═O)— (unsubstituted lower alkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted aryloxy;

aryloxy substituted with a group independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxy and amino;

mercapto;

unsubstituted alkylthio;

unsubstituted arylthio;

arylthio substituted with one or more groups independently selected from the group consisting of halo, hydroxy and amino;

lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted lower alkoxy;

lower alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″, unsubstituted aryl or —NR13R14;

trihalomethyl;

unsubstituted alkenyl;

unsubstituted alkynyl;

unsubstituted aryl;

aryl substituted with one or more groups independently selected from the groups consisting of unsubstituted lower alkyl or lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted heteroalicyclic;

heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, —C(═O)H, —C(═O)— (unsubstituted lower alkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted aryloxy;

aryloxy substituted with a one or more groups independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxy and amino;

mercapto;

unsubstituted alkylthio;

unsubstituted arylthio;

arylthio substituted with one or more groups independently selected from the group consisting of halo, hydroxy or amino;

S-sulfonamido;

—C(═O)OR″;

R″C(═O)O—;

hydroxy;

cyano;

nitro;

halo;

C-amido;

N-amido;

amino; and,

—NR13R14, wherein

R13 is hydrogen and R14 is unsubstituted lower alkyl is another presently preferred embodiment of this invention.

A still further presently preferred embodiment of this invention is a compound in which:

R10, R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, —(CH2)C(═O)OR″, —(CH2)rC(═O)O−M+, halo, hydroxy, alkoxy, R″C(═O)O—, —C(═O)OR″, —C(═O)O−M+, amino, C-amido, N-amido, nitro and —NR13R14 is also a presently preferred embodiment of this invention.

A compound having the structural features in the paragraph immediately above wherein at least one of R10, R11 or R12 is selected from the group consisting of —C(═O)OR″, —C(═O)O−M+, —(CH2)rC(═O)OR″ and —(CH2)rC(═O)O−M+ is a presently preferred embodiment of this invention.

It is also a presently preferred embodiment of this invention that, in a compound having the structural features described in the paragraph immediately above this one, r of the —(CH2)rC(═O)OR″ or —(CH2)rC(═O)O− M+ group is 1 or 2.

Representative compounds of this invention are shown in Table 1. The compounds shown are presented by way of example only and are not to be construed as limiting the scope of this invention in any manner whatsoever.

2. BRIEF DESCRIPTION OF THE TABLES

1. Table 1 shows the chemical structures of exemplary compounds of this invention. The compound numbers correspond to the compound numbers in the Examples section, below. That is, the synthesis of compound 1 in Table 1 is Example 1 in the Examples section. These compounds are presented as examples only and are not to be construed as limiting the scope of this invention in any manner whatsoever.

2. Table 2 shows the results of biological assays of exemplary compounds of this invention. As above, the compound numbers is Table 2 correspond to the compound numbers in Table 1. The bioassays used are described in detail below. The results are given in terms of IC50, the micromolar (μM) concentration of the compound being tested which effects a 50% change in the activity of a target PTK compared to the activity of the PTK in a control in which no compound of this invention is present. Specifically, the results shown indicate the concentration of the test compound needed to effect a 50% inhibition of the activity of the target PTK observed in the absence of a compound of this invention.

3. Table 3 shows the results of in vivo tests using the A375 sc cell line in the animal xenograft model described below. Compound 13 has the chemical structure shown in Table 1.

TABLE 1

Compound No.:

Structure

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

3. THE BIOCHEMISTRY

In yet another embodiment, this invention relates to a method for the modulation of the catalytic activity of PKs by contacting a PK with a compound of this invention or a physiologically acceptable salt or prodrug thereof.

A further embodiment of this invention is a method for identifying a compound which modulates the activity of a PK whichmethod consists of contacting a cell which the PK of interest with a compound and monitoring the effect of the compound on the cell.

By “PK” is meant RTKs, CTKs and STKs; i.e., the modulation of RTK, CTK and STK catalyzed signaling processes are contemplated by this invention.

The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by, practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “modulation” or “modulating” refers to the alteration of the catalytic activity of RTKs, CTKs and STKs. In particular, modulating refers to the activation of the catalytic activity of RTKS, CTKs and STKS, preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to which the RTK, CTK or STK is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and STKs.

The term “catalytic activity” as used herein refers to the rate of phosphorylation of tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation of serine and threonine under the influence, direct or indirect, of STKs.

The term “contacting” as used herein refers to bringing a compound of this invention and a target PK together in such a manner that the compound can affect the catalytic activity of the PK, either directly; i.e., by interacting with the kinase itself, or indirectly; i.e., by interacting with another molecule on which the catalytic activity of the kinase is dependent. Such “contacting” can be accomplished in a test tube, a petri dish or the like. In a test tube, contacting may involve only a compound and a PK of interest or it may involve whole cells. Cells may also be maintained or grown in cell culture dishes and contacted with the compound in that environment. In this context, the ability of a particular compound to affect a PK related disorder; i.e., the IC50, of the compound, defined below, can be determined before use of the compounds in vivo with more complex living organisms is attempted. For cells outside the organism, multiple methods exist, and are well-known to those skilled in the art, to get the PKs in contact with the compounds including, but not limited to, direct cell microinjection and numerous transmembrane carrier techniques.

By “monitoring” or “observing” is meant detecting the effect of contacting a compound with a cell expressing a particular PK.

The detected effect can be a change in cell phenotype, in the catalytic activity of a PK or a change in the interaction of a PK with a natural binding partner.

“Cell phenotype” refers to the outward appearance of a cell or tissue or the biological function of the cell or tissue. Examples, without limitation, of a cell phenotype is cell size, cell growth, cell differentiation, cell proliferation, cell survival, apoptosis and nutrient uptake and use. Such phenotypic characteristics are detectable by techniques well-known in the art.

A “natural binding partner” refers to a polypeptide that binds to a particular PK in a cell. Natural binding partners can plan a role in propagating a signal in a PK-mediated signal transduction process. A change in the interaction of the natural binding partner with the PK can manifest itself as an increased or decreased concentration of the PK/natural binding partner complex resulting in a detectable change in the ability of the PK to mediate signal transduction.

RTK mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects on the extracellular microenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron 9:303-391.

It has been shown that tyrosine phosphorylation sites on growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423; Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678. Several intracellular substrate proteins that associate with RTKs have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993, Cell, 72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993, Cell, 72:767-778. These observations suggest that the function of each RTK is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors as well as differentiation factor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process which initiates DNA synthesis and subsequent mitosis leading to cell proliferation.

Thus, PK signal transduction results in, among other responses, cell proliferation, differentiation, growth and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy and other disorders related to uncontrolled angiogenesis and/or vasculogenesis.

A precise understanding of the mechanism by which the compounds of this invention inhibit PKs is not required in order to practice the present invention. However, while not hereby being bound to any particular mechanism or theory, it is believed that the compounds interact with the amino acids in the catalytic region of PKs. PKs typically possess a bi-lobate structure wherein ATP appears to bind in the cleft between the two lobes in a region where the amino acids are conserved among PKs. Inhibitors of PKs are believed to bind by non-covalent interactions such as hydrogen bonding, van der Waals forces and ionic interactions in the same general region where the aforesaid ATP binds to the PKs. More specifically, it is thought that the 2-indolinone component of the compounds of this invention binds in the general space normally occupied by the adenine ring of ATP. Specificity of a particular molecule for a particular PK may then arise as the result of additional interactions between the various substituents on the 2-indolinone core and the amino acid domains specific to particular PKs. Thus, different indolinone substituents may contribute to preferential binding to particular PKs. The ability to select compounds active at different ATP (or other nucleotide) binding sites makes the compounds of this invention useful for targeting any protein with such a site; i.e., not only PKs but protein phosphatases as well. The compounds disclosed herein may thus have utility as in vitro assays for such proteins as well as exhibiting in vivo therapeutic effects through interaction with such proteins.

In another aspect, the protein kinase, the catalytic activity of which is modulated by contact with a compound of this invention, is a protein tyrosine kinase, more particularly, a receptor protein tyrosine kinase. Among the receptor protein tyrosine kinases whose catalytic activity can be modulated with a compound of this invention, or salt thereof, are, without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.

The protein tyrosine kinase whose catalytic activity is modulated by contact with a compound of this invention, or a salt or a prodrug thereof, can also be a non-receptor or cellular protein tyrosine kinase (CTK). Thus, the catalytic activity of CTKs such as, without limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compound or salt of this invention.

Still another group of PKs which may have their catalytic activity modulated by contact with a compound of this invention are the serine-threonine protein kinases such as, without limitation, CDK2 and Raf.

In another aspect, this invention relates to a method for treating or preventing a PK related disorder by administering a therapeutically effective amount of a compound of this invention, or a salt or a prodrug thereof, to an organism.

In a further aspect, this invention relates to a method for treating or preventing a PK related disorder administering a therapeutically effective amount of a pharmacological composition of a compound of this invention, or a salt or prodrug thereof, to an organism.

As used herein, “PK related disorder,” “PK driven disorder,” and “abnormal PK activity” all refer to a condition characterized by inappropriate; i.e., under or, more commonly, over, PK catalytic activity, where the particular PK can be an RTK, a CTK or an STK. Inappropriate catalytic activity can arise as the result of either: (1) PK expression in cells which normally do not express PKs; (2) increased PK expression leading to unwanted cell proliferation, differentiation and/or growth; or, (3) decreased PK expression leading to unwanted reductions in cell proliferation, differentiation and/or growth. Over-activity of PKs refers to either amplification of the gene encoding a particular PK or production of a level of PK activity which can correlate with a cell proliferation, differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of one or more of the symptoms of the cellular disorder increases). Underactivity is, of course, the converse, wherein the severity of one or more symptoms of a cellular disorder increase as the level of the PK decreases.

As used herein, the terms “prevent”, “preventing” and “prevention” refer to a method for barring an organism from acquiring a PK mediated cellular disorder in the first place.

As used herein, the terms “treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a PK mediated cellular disorder and/or its attendant symptoms. With regard particularly to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced.

The term “organism” refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukariotic cell or as complex as a mammal, including a human being.

The term “therapeutically effective amount” as used herein refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of the tumor; (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis; (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth; and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.

This invention is therefore directed to compounds which modulate PK signal transduction by affecting the enzymatic activity of RTKs, CTKs and/or STKs, thereby interfering with the signals transduced by such proteins. More particularly, the present invention is directed to compounds which modulate RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinomas, sarcomas, including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Treatment or prevention of non-solid tumor cancers such as leukemia are also contemplated by this invention. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders related to unregulated PK activity that the compounds described herein may be useful in preventing, treating and studying, are cell proliferative disorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders, which may be prevented, treated or further studied by the present invention include cancer, blood vessel proliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (spreading of blood vessels). While vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration, they also play a pivotal role in cancer development where they result in the formation of new capillaries needed to keep a tumor alive. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness.

Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, may also be treated or prevented by the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Another fibrotic disorder is atherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis as well such disorders as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney International 43:47Sa54S.

As noted previously, PKs have been associated with cell proliferative disorders. Thus it is not surprising that some members of the RTK family have been associated with the development of cancer. Some of these receptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) are over-expressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor over-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci., 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have been demonstrated. For example, EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated.with breast, ovarian, gastric, lung, pancreas and bladder cancer. PDGFR has been associated with glioblastoma and melanoma as well as lung, ovarian and prostate cancer. The RTK c-met has been associated with malignant tumor formation. For example, c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas. Additionally c-met has been linked to leukemia. Over-expression of the c-met gene has also been detected in patients with Hodgkins disease and Burkitts disease.

Flk/KDR has likewise been associated with a broad spectrum of tumors including, without limitation, mammary, ovarian and lung tumors as well as gliomas such as glioblastoma.

IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). In addition, IGF-I, while integrally involved in the normal growth and differentiation of the nervous system, also appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The importance of IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stem cells of the bone marrow)) are stimulated to grow by IGF-I. Goldring and Goldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series of recent publications, Baserga suggests that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga, 1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol., 14:4588-4595.

The association between abnormal PK activity and disease is not restricted to cancer. For example, RTKs have been associated with diseases such as psoriasis, diabetes mellitus, endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease, epidermal hyperproliferation, neuro-degenerative diseases, age-related macular degeneration and hemangiomas. For instance, EGFR is indicated in corneal and dermal wound healing. Defects in Insulin-R and IGF-1R are indicated in type-II diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., 1994, DN&P, 7:334-339.

As noted previously, not only RTKs but CTKs as well including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolen et al., 1992, FASEB J., 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus could be expected, and have been shown, to be involved in many PTK-mediated disorders to which the present invention is directed. For example, mutated src (v-src) has been demonstrated to be an oncoprotein (pp60v-src) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60c-src transmits oncogenic signals of many receptors. Over-expression of EGFR or HER2/neu in tumors leads to the constitutive activation of pp60cXsrc, which is characteristic of malignant cells but absent in normal cells. On the other hand, mice deficient in the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders.

Similarly, Zap70 has been connected with T-cell signaling which may have implications in autoimmune disorders.

STKs have been associated with inflamation, autoimmune disease, immunoresponses, and hyperproliferation disorders such as restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, the compounds of this invention may provide an effective method of preventing such embryo implantation and thereby be useful as birth control agents.

Finally, both RTKs and CTKs are currently suspected as being involved in hyperimmune disorders. Thus, it is an aspect of this invention that protein kinase related cancers such as, without limitation, squamous cell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, small-cell lung cancer, glioma, colorectal cancer, genitourinary cancer and gasterointestinal cancer may be treated or prevented by administration to an organism of a therapeutically effective amount of a compound of this invention.

In a further aspect of this invention, non-cancer protein kinase related diorders such as, without limitation, diabetes, autoimmune disorder, immunological disorder, hyperproliferative disorder, restenosis, fibrosis, psoriasis, von Hippel-Lindau disease, osteoarthritis, rheumatoid arthritis, angiogenesis, inflammatory disorder and cardiovascular disease, may also be treated or prevented by the administration of a therapeutically effective amount of a compound of this invention to an organism.

Tables 2 and 3 show the activity of representative compounds of this invention against several of the above-described RTKs. Neither the compounds shown, the levels of activity indicated nor the specific RTKs affected are to be construed as limiting the scope of this invention in any manner whatsoever.

TABLE 2

bio

EGFR

bio

cell

VEGF

Compound

(Page 5)

PDGFR

Pyk2

cell PDGFR

bio Flk

EGFR

cell IGF

bio FGF

bio SRC

HUVEC

1

>100

>100

>100

54.2

18.4

DNH

DNH

2

>100

>100

DNH

>100

8.2

DNH

DNH

3

>100

86.9

DNH(56%

30.2

2.6

DNH

DNH

Inh)

4

>100

>100

DNH

>100

DNH

DNH

5

>100

50.8

79.3

10.9

3.1

DNH

>100

6

>100

5.5

8.1

2.5

96.7% Inh

>100

7

>100

>100

DNH

>100

7.1

DNH

>100

8

>100

>100

DNH

>100

4.3

DNH

9

>100

>100

>100

2.9

DNH

10

>100

63.7

>100

1.4

>100

>100

56.8

>100

0.33

11

>100

1.5

9.3

>100

0.02

>100

>100

0.2

0.05

12

>25

>25

DNH

>25

>25

>25

>25

>25

13

>100

1.0, 3.2

4.5

>100

>100

>100

14

DNH = did not hit in primary assay

TABLE 3

Average

Tumor

Volume

%

Treatment

(mm3)

inhibition

P value

%

Vehicle

756.1

—

—

40

Compound 13 @ 200 mg/kg/day

278.8

63.1

>0.05

40

Compound 13 @ 100 mg/kg/day

324.4

57.1

>0.05

10

Compound 13 @ 50 mg/kg/day

569.9

24.6

>0.05

20

4. PHARMACOLOGICAL COMPOSITIONS AND THERAPEUTIC APPLICATIONS

A compound of the present invention, a prodrug thereto or a physiologically acceptable salt of either the compound or its prodrug, can be administered as such to a human patient or in pharmacological compositions in which the foregoing materials are mixed with suitable carriers or excipient(s). Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

Routes of Administration.

As used herein, “administer” or “administration” refers to the delivery of a compound, salt or prodrug of the present invention or of a pharamacological composition containing a compound, salt or prodrug of this invention to an organism for the purpose of prevention or treatment of a PK-related disorder.

Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.

Composition/Formulation.

Pharmacological compositions of the present invention may be manufactured by processes well known in the art; e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmacological compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.

For transmucosal administration, penetrants appropriate to the barrier to be permeated ate used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmacological compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added in these formulations, also.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may also be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating materials such as suspending, stabilizing and/or dispersing agents.

Pharmacological compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers and/or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. A compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharamcologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt.

The pharmacological compositions herein also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided as physiologically acceptable salts wherein the claimed compound may form the negatively or the positively charged species. Examples of salts in which the compound forms the positively charged moiety include, without limitation, quaternary ammonium (defined elsewhere herein), salts such as the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention which has reacted with the appropriate acid. Salts in which a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g. sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide (Ca(OH)2), etc.).

Dosage.

Pharmacological compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose; i.e., the modulation of PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PK activity). Such information can then be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 and the LD50 (both of which are discussed elsewhere herein) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active specie which are sufficient to maintain the kinase modulating effects. These plasma levels are referred to as minimal effective concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of a kinase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.

In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Packaging.

The compositions may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or of human or veterinary administration. Such notice, for example, may be of the labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.

5. SYNTHESIS

The compounds of this invention, as well as the precursor 2-oxindoles and cycloketones, may be synthesized using the procedure described below. Other approaches to the synthesis of the compounds and/or precursors to the compounds of this invention may become apparent to those skilled in the art based on the disclosures herein. Such alternate procedures are within the scope and spirit of this invention.

General Synthetic Procedure.

One to three equivalents of the appropriate 2-oxindole and one equivalent of the appropriate cycloketone are mixed together in water or an organic solvent such as, without limitation, C1-C10 alcohols (methanol, ethanol, octanol, etc.), ethylene glycol, cellosolve, diethyl ether, dichloromethane, chloroform, carbon tetrachloride, benzene, toluene, xylene, tetrahydrofuran, acetonitrile, dioxane, dimethylsulfoxide, dimethylformamide, dimethylacetamide, 1-methyl-2-pyrrolidine, pyridine and the like. Preferably the solvent is an organic solvent selected from the group consisting of dimethylsulfoxide, dimethylformamide, dimethylacetamide and N-methylpyrrolidine. Most preferably, the solvent is selected from dimethylformamide and dimethylacetamide. A molar excess of an inorganic or organic base is added. The base may be, without limitation, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium methoxide, sodium ethoxide, potassium t-butoxide, triethylamine, triethanolamine, piperidine, morpholine, pyrrolidine and the like. Preferably, the base is an organic base selected from the group consisting of triethylamine, piperidine, morpholine and pyrrolidine. Most preferably, the base is piperidine. The resulting solution or mixture is stirred at from about 20° C. to about 200° C. for from about 0.5 hour to about 100 hours at atmospheric pressure or in a sealed tube. The temperature is preferably about 90° C. to about 175° C., most preferably about 110° C. to about 150° C. The reaction time is preferably 1 hour to about 72 hours. The mixture is then brought to room temperature. Dilute aqueous inorganic acid (for example, without limitation, 1N hydrochloric acid or 1N sulfuric acid) is then added in an amount sufficient to neutralize the base. The resulting mixture is extracted with a water insoluble organic solvent such as without limitation, methylene chloride, diethyl ether, hexane, ethyl acetate, benzene or mixtures of solvents such as ethyl acetate/hexane. Preferably, the organic solvent is ethyl acetate. The organic solvent layer is separated, dried and evaporated to give the compound of this invention.

SPECIFIC SYNTHESES

The exemplary syntheses which follow are not to be construed as limiting the scope of this invention in any manner.

EXAMPLE 13-(3-Methylindanl-ylidene)-1,3-dihydroindol-2-one

A mixture of 0.5 g of 3-methyl-1-indanone, 1.37 g oxindole and 5 ml piperidine in 3 ml of dimethylforamide was heated in a sealed tube at 130° C. overnight to yield a reddish-brown suspension. The mixture was added to 1N hydrochloric acid solution and extracted with ethyl acetate. The organic layer was washed with brine, dried with magnesium sulfate and concentrated. Chromatography afforded 120 mg of 3-(3-methylindan-1-ylidene)-1,3-dihydroindol-2-one as an orange solid.

A mixture of 0.5 g 4-methyl-1-indanone, 1.37 g oxindole and 3 ml piperidine in 5 ml dimethylforamide was heated in a sealed tube at 130° C. for 3 days to yield a reddish-black suspension. The mixture was added to 1N hydrochloric acid solution and extracted with ethyl acetate. The organic layer was washed with brine dried over magnesium sulfate and concentrated. The resulting solid was precipitated from ethyl acetate/hexanes (2×) followed by methylene chloride/hexanes to afford 400 mg of 3-(4-methyl-indan-1-ylidene)-1,3-dihydroindol-2-one as a yellow solid.

A mixture of 0.5 g 5-methoxy-1-indanone, 1.23 g oxindole and 2.7 ml piperidine in 4 ml dimethylforamide was heated in a sealed tube at 130° C. for 3 days to yield a greenish-black suspension. The mixture was added to 1N hydrochloric acid solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. Chromatography afforded 20 mg of 3-(5-methoxyindan-1-ylidene)-1,3-dihydroindol-2-one as a brown solid.

A mixture of 0.5 g 6-methoxy-1-indanone, 1.23 g oxindole and 2.7 ml piperidine in 4 ml of dimethylforamide was heated in a sealed tube 130° C. for 3 days to yield a greenish-black suspension. The mixture was added to 1N hydrochloric acid solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The resulting solid was precipitated from methylene chloride/hexanes (3×) to afford 290 mg 3-(6-methoxyindan-1-ylidene)-1,3-dihydroindol-2-one as a yellowish-brown solid.

A mixture of 0.5 g 5-dimethylamino-1-indanone, 1.1 g oxindole and 2.5 ml piperidine in 4 ml dimethylforamide was heated in a sealed tube at 130° C. for 12 hours to yield a reddish-black suspension. The mixture was added to 1N hydrochloric acid solution and extracted with ethyl acetate. The organic layer was washed with brine, dried with magnesium sulfate and concentrated. Chromatography afforded 35 mg of 3-(5-dimethylamino-indan-1-ylidene)-1,3-dihydroindol-2-one as a brown solid.

A mixture of 0.5 g 5-amino-1-indanone, 1.3 g oxindole and 3 ml piperidine in 5 ml of dimethylforamide was heated in a sealed tube at 130° C. for 3 days to yield a reddish-black suspension. The mixture was added to 1N hydrochloric acid and extracted with ethyl acetate. The organic layer was washed with brine, dried with magnesium sulfate and concentrated. Chromatography afforded 45 mg of 3-(5-aminoindan-1-ylidene)-1,3-dihydroindol-2-one as a brown solid.

A mixture of 500 mg 5-chloro-1-indanone, 1.33 g oxindole and 0.5 ml piperidine in 3 ml dimethylforamide was heated in a sealed tube at 95° C. overnight. Water was added to the reaction mixture to yield.an oily solid. The mixture was sonicated for a few minutes then decanted. This washing procedure was repeated several times. The solid was then filtered and washed with ethyl acetate/hexanes (1:3) to yield 172 mg of 3-(5-chloroindan-1-ylidene)-1,3-dihydroindol-2-one as a mustard colored solid.

A mixture of 0.5 g 5-fluoro-1-indanone, 13 g oxindole and 1 ml piperidine in 4 ml dimethylforamide was heated in a sealed tube at 125° C. for 2.5 hours. The reaction mixture was poured into water and the aqueous layer was decanted. Ethyl acetate was then added slowly until a solid formed. The solid was filtered and washed with ethyl acetate/hexanes to yield a curry colored solid. The solid was then further purified by washing with dimethylforamide/acetone to give 122 mg of 3-(5-piperidin-1-yl-indan-1-ylidene)-1,3-dihydroindol-2-one as an orange solid.

A reaction mixture of 0.422 g 5-bromoindanone, 1.3 g oxindole and 0.9 ml piperidine in 3 ml of dimethylforamide was heated in a sealed tube at 110° C. overnight. The reaction mixture was poured into ice water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated. The black residue was chromatographed on a silica gel column, eluting with ethyl acetate and hexane to give 73.0 mg of 3-(5-bromoindan-1-ylidene)-1,3-dihydroindol-2-one as a yellow solid.

A mixture of 0.4 g indan-2-one, 1.33 g oxindole and 0.5 ml piperidine in 3 ml dimethylforamide was heated in a sealed tube at 130° C. for 60 hours. Enough water was added to cause precipitation of an oily solid. The mixture was decanted and the remaining oily solid heated with 5 ml of ethanol using a heat gun. Upon cooling, the solid was filtered off. The solid was slurried with 3 ml of ethanol and filtered to give 160 mg of 3-indan-1-ylidene-1,3-dihydroindol-2-one as an orange solid.

A mixture of 0.66 g 5-methoxyindan-3-one acetic acid, 2.4 g oxindole and 1.0 ml piperidine in 3 ml of dimethylformamide was heated in a sealed tube at 130° C. for 14 hours. Three ml of 6N hydrochloric acid was added to the cooled reaction mixture followed by 3 ml of water. The supernatant was then decanted. The oily residue was heated in 5 ml of ethanol until it dissolved, the solution was then cooled and water was added. After standing overnight, a solid formed which was filtered and washed with ethanol. The solid was slurried in 4 ml of ethyl acetate at 70° C. for 45 minutes, filtered and suctioned dried to give 300 mg of [6-methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)-indan-1-yl]-acetic acid.

A mixture of 0.6 g 5,6-dimethoxy-1-indanone, 1.04 g oxindole and 2.3 ml piperidine in 4 ml dimethylformamide was heated in a sealed tube at 130° C. for 12 hours to yield an orange suspension. The mixture was added to 1N hydrochloric acid in ethanol and the solids which formed were filtered and rinsed with water and ethanol. The solid was slurried in ethanol and filtered to yield 200 mg of 3-(5,6-dimethoxyindan-1-ylidene)-1,3-dihydroindol-2-one.

A suspension of 8 g [6-methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)indan-1-yl]-acetic acid in 60 ml of water was added to 0.9 g of sodium hydroxide in 10 ml of water. The mixture was stirred at room temperature for 30 minutes and filtered. The filtrate was frozen and lyophilized to give 8 g of [6-methoxy-3-(2-oxo-1,2-dihydroindol-3-ylidene)-indan-1-yl]-acetic acid sodium salt.

A mixture of 680 mg 1,5,6,7-tetrahydro-4H-indol-4-one and 1.33 g oxindole in 3 ml dimethylforamide was heated at 140° C. for 50 hours. The mixture was diluted with water and extracted with ethyl acetate. The organic extracts were washed with water and then brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified on a silica gel column using hexanes/ethyl acetate as the eluent to provide 150 mg of 1′,5′,6′,7′-tetrahydro-1H-[3,4′]biindolyliden-2-one as a yellow solid.

It will be appreciated that, in any given series of compounds, a spectrum of biological activities will be obtained. In its preferred embodiments, this invention relates to novel geometrically restricted 2-indolinones demonstrating the ability to modulate RTK, CTK, and STK activity. The following assays are employed to select those compounds demonstrating the optimal degree of the desired activity.

Assay Procedures.

The following in vitro assays may be used to determine the level of activity and effect of the different compounds of the present invention on one or more of the PKs. Similar assays can be designed along the same lines for any PK using techniques well known in the art.

The cellular/catalytic assays described herein are performed in an ELISA format. The.general procedure is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. The cells are lysed and the lysate is transferred to the wells of an ELISA plate previously coated with a specific antibody recognizing the substrate of the enzymatic phosphorylation reaction. Non-substrate components of the cell lysate are washed away and the amount of phosphorylation on the substrate is detected with an antibody specifically recognizing phosphotyrosine compared with control cells that were not contacted with a test compound.

The cellular/biologic assays described herein measure the amount of DNA made in response to activation of a test kinase, which is a general measure of a proliferative response. The general procedure for this assay is as follows: a compound is introduced to cells expressing the test kinase, either naturally or recombinantly, for a selected period of time after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. After incubation at least overnight, a DNA labeling reagent such as Bromodeoxyuridine (BrdU) or 3H-thymidine is added. The amount of labeled DNA is detected with either an anti-BrdU antibody or by measuring radioactivity and is compared to control cells not contacted with a test compound.

Cellular/Catalytic Assays

Enzyme linked immunosorbent assays (ELISA) may be used to detect and measure the presence of PK activity. The ELISA may be conducted according to known protocols which are described in, for example, Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual of Clinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C.

The disclosed protocol may be adapted for determining activity with respect to a specific PK. That is, the preferred protocols for conducting the ELISA experiments for specific PKs is provided below. However, adaptation of these protocols for determining a compound's activity for other members of the RTK family, as well as for CTKs and STKs, is well within the scope of knowledge of those skilled in the art.

FLK-1 Assay

An ELISA assay is conducted to measure the kinase activity of the FLK-1 receptor and more specifically, the inhibition or activation of TK activity on the FLK-1 receptor. Specifically, the following assay can be conducted to measure kinase activity of the FLK-1 receptor in cells genetically engineered to express Flk-1.

9. Add 18 μl of 1:20 diluted compound dilution (from step 7) to each well plus the 1:20 dimethylsulfoxide dilution to the control wells (+/−VEGF), for a final dilution of 1:200 after cell stimulation. Final dimethylsulfoxide is 0.5%. Incubate the plate at 37° C., 5% CO2 for two hours.

25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final concentration to stop the color development reaction. Shake 1 minute at room temperature. Remove bubbles with slow stream of air and read the ELISA plate in an ELISA plate reader at 410 nm.

HER-2 ELISA

EGF Receptor-HER2 Chimeric Receptor Assay in Whole Cells.

HER2 kinase activity in whole EGFR-NIH3T3 cells are measured as described below:

1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 μg per well in PBS, 100 μl final volume/well, and store overnight at 4° C. Coated plates are good for up to 10 days when stored at 4° C.

1. Check seeded cells for contamination using an inverted microscope. Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer 5 μl to a TBST well for a final drug dilution of 1:200 and a final DMSO concentration of 1%. Control wells receive DMSO alone. Incubate in 5% CO2 at 37° C. for two hours.

5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above.

6. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour.

7. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST).

11. The maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.

PDGF-R Assay

All cell culture media, glutamine, and fetal bovine serum can be purchased from Gibco Life Technologies (Grand Island, N.Y.) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% CO2 at 37° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco).

For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU) are grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in 0.5% serum-containing medium, cells are changed to serum-free medium and treated with test compound for 2 hr in a 5% CO2, 37° C. incubator. Cells are then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na3VO4, 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates (0.5 mg/well in PBS) are transferred to ELISA plates previously coated with receptor-specific antibody and which had been blocked with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100) at room temperature for 30 min. Lysates are incubated with shaking for 1 hour at room temperature. The plates are washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody is removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody is added to the ELISA plate for 30 min at room temperature followed by rinsing with TBST four more times. ABTS (100 mM citric acid, 250 mM Na2HPO4 and 0.5 mg/mL 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H2O2 (1.2 mL 30% H2O2 to 10 ml ABTS) is added to the ELISA plates to start color development. Absorbance at 410 nm with a reference wavelength of 630 nm is recorded about 15 to 30 min after ABTS addition.

IGF-1 RECEPTOR

The following protocol may be used to measure phosphotyrosine level on IGF-1 receptor, which indicates IGF-1 receptor tyrosine kinase activity.

Materials and Reagents.

a. The cell line used in this assay is 3T3/IGF-1R, a cell line genetically engineered to overexpresses IGF-1 receptor.

b. NIH3T3/IGF-1R is grown in an incubator with 5% CO2 at 37° C. The growth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.

ABTS solution should be kept in dark and 4° C. The solution should be discarded when it turns green.

o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4° C.

Procedure.

All the following steps are conducted at room temperature unless specifically indicated otherwise. All ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST rinse. Pat plate dry with paper towels.

2. Remove the coating solution, and replace with 100 μl Blocking Buffer, and shake for 30 minutes. Remove the blocking buffer and wash the plate just before adding lysate.

Assay Procedures:

1. The drugs are tested under serum-free condition.

2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-propylene plate, and transfer 10 μl/well of this solution to the cells to achieve final drug dilution 1:100, and final DMSO concentration of 1.0%. Incubate the cells in 5% CO2 at 37° C. for 2 hours.

3. Prepare fresh cell lysis buffer (HNTG*)

HNTG

2 ml

EDTA

0.1 ml

Na3VO4

0.1 ml

Na4(P2O7)

0.1 ml

H2O

7.3 ml

4. After drug incubation for two hours, transfer 10 μl/well of 200 nM IGF-1 Ligand in PBS to the cells (Final Conc. is 20 nM), and incubate at 5% CO2 at 37° C. for 10 minutes.

5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Look at cells under microscope to see if they are adequately lysed.

6. Use a 12-channel pipette to scrape the cells from the plate, and homogenize the lysate by repeated aspiration and dispensing. Transfer all the lysate to the antibody coated ELISA plate, and shake for 1 hour.

1. Coat ELISA plates (Corning, 96-well, Cat. #25805-96) with 05-101 antibody at 0.5 μg per well in PBS, 150 μl final volume/well, and store overnight at 4° C. Coated plates are good for up to 10 days when stored at 4° C.

1. Check seeded cells for contamination using an inverted microscope. Dilute test compounds stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer 5 μl to a test well for a test compounds drug dilution of 1:200 and a final DMSO concentration of 1%. Control wells receive DMSO alone. Incubate in 5% CO2 at 37° C. for one hour.

5. After two hours incubation with drug, add prepared EGF ligand to cells, 10 μl per well, to yield a final concentration of 25 nM. Control wells receive DMEM alone. Incubate, shaking, at room temperature, for 5 minutes.

6. Remove test compound, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above.

7. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour.

8. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST).

12. The maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls.

r. Vectastain ELITE ABC reagent: To prepare 14 ml of working reagent, add 1 drop of reagent A to 15 ml TBST and invert tube several times to mix. Then add 1 drop of reagent B. Put tube on orbital shaker at room temperature and mix for 30 minutes.

c. GST-ζ: DNA encoding for GST-ζ fusion protein for expression in bacteria obtained from Arthur Weiss of the Howard Hughes Medical Institute at the University of California, San Francisco. Transformed bacteria are grown overnight while shaking at 25° C. GST-ζ is purified by glutathione affinity chromatography, Pharmacia, Alameda, Calif.

12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilution in 100 μl of TBST for 30 min. at room temperature.

13. Wash the wells 5× with TBST.

14. Develop with Turbo TMB.

Assay Measuring Phosphorylating Function of RAF.

The following assay reports the amount of RAF-catalyzed phosphorylation of its target protein MEK as well as MEK's target MAPK. The RAF gene sequence is described in Bonner et al., 1985, Molec. Cell. Biol., 5:1400-1407, and is readily accessible in multiple gene sequence data banks. Construction of the nucleic acid vector and cell lines utilized for this portion of the invention are fully described in Morrison et al., 1988, Proc. Natl. Acad. Sci. USA, 85:8855-8859.

3. Remove blocking solution and wash four times with wash buffer. Pat the plate on a paper towel to remove excess liquid.

4. Add 1 mg of antibody specific for RAF-1 to each well and incubate for 1 hour. Wash as described in step 3.

5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to 10 mg/100 mL. Add 10 mg of diluted lysate to the wells and incubate for 1 hour. Shake the plate during incubation. Negative controls receive no lysate. Lysates from RAS/RAF infected Sf9 insect cells are prepared after cells are infected with recombinant baculoviruses at a MOI of 5 for each virus, and harvested 48 hours later. The cells are washed once with PBS and lysed in RIPA buffer. Insoluble material is removed by centrifugation (5 min at 10,000× g). Aliquots of lysates are frozen in dry ice/ethanol and stored at −80° C. until use.

6. Remove non-bound material and wash as outlined above (step 3).

7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjust the volume to 40 ml with kinase buffer. Methods for purifying T-MEK and MAPK from cell extracts are provided herein by example.

9. Start the kinase reaction by addition of 5 ml ATP mix; Shake the plates on an ELISA plate shaker during incubation.

10. Stop the kinase reaction after 60 min by addition of 30 mL stop solution to each well.

11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate harvester. Harvest and wash the filter with the filter wash solution according to the manufacturer's recommendation. Dry the filter mats. Seal the filter mats and place them in the holder. Insert the holder into radioactive detection apparatus and quantify the radioactive phosphorous on the filter mats.

Alternatively, 40 mL aliquots from individual wells of the assay plate can be transferred to the corresponding positions on the phosphocellulose filter mat. After air drying the filters, put the filters in a tray. Gently rock the tray, changing the wash solution at 15 min intervals for 1 hour. Air-dry the filter mats. Seal the filter mats and place them in a holder suitable for measuring the radioactive phosphorous in the samples. Insert the holder into a detection device and quantify the radioactive phosphorous on the filter mats.

CDK2/Cyclin A—Inhibition Assay

This assay analyzes the protein kinase activity of CDK2 in exogenous substrate.

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO2.

(2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

(5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.

(1) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO2.

(2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

(5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.

(2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

(5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.

(2) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.

(5) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.

Mix first two ingredients in about 900 ml dH2O, adjust pH to 4.0 with phosphoric acid. Add ABTS, cover, let sit about 0.5 hr., filter. The solution should be kept in the dark at 4° C. until ready to use.

12. Add 15 μl phosphorylation mix directly to all wells except negative control well which does not receive ATP/MnCl2 (final well volume should be approximately 150 μl with 3 μM ATP/5 mM MnCl2 final concentration in each well.) Incubate 5 minutes while shaking.

13. After 5 minutes, stop reaction by adding 16.5 μl of 200 mM EDTA (pH 8.0) to each well, shaking continuously. After the EDTA has been added, shake for 1 min.

This assay measures in vitro kinase activity of the Myc-GyrB-FGFR fusion protein using ELISA.

Materials and Reagents

1. HNTG

5x Stock

Amount

1x Working

Reagent

MW

Concentration

per L

Concentration

HEPES

238.3

100 mM

23.83

g

20 mM

NaCl

58.44

750 mM

43.83

g

150 mM

Glycerol

NA

50%

500

ml

10%

Triton X-100

NA

5%

10

ml

1.0%

To make a liter of 5×stock solution, dissolve HEPES and NaCl in about 350 ml dH2O, adjust pH to 7.2 with HCl or NaOH (depending on the HEPES that is used), add glycerol, Triton X-100 and then dH2O to volume.

2. Wash the cells with about 35 ml assay medium in the 50 ml sterile centrifuge tube by adding the assay medium, centrifuge for 10 minutes at approximately 200×g, aspirate the supernatant, and resuspend with 35 ml D-PBS. Repeat the wash two more times with D-PBS, resuspend the cells in about 1 ml assay medium/15 cm2 of tissue culture flask. Assay medium consists of F12K medium (Gibco BRL; catalogue no. 21127-014) and 0.5% heat-inactivated fetal bovine serum. Count the cells with a Coulter Counter® (Coulter Electronics, Inc.) and add assay medium to the cells to obtain a concentration of 0.8-1.0×105 cells/ml.

1. Make up two-fold test compound titrations in separate 96-well plates, generally 50 μM on down to 0 μM. Use the same assay medium as mentioned in day 0, step 2 above. Titrations are made by adding 90 μl/well of test compound at 200 μM (4× the final well concentration) to the top well of a particular plate column. Since the stock test compound is usually 20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovine serum) is used as diluent for the test compound titrations in order to dilute the test compound but keep the DMSO concentration constant. Add this diluent to the remaining wells in the column at 60 μl/well. Take 60 μl from the 120 μl of 200 μM test compound dilution in the top well of the column and mix with the 60 μl in the second well of the column. Take 60 μl from this well and mix with the 60 μl in the third well of the column, and so on until two-fold titrations are completed. When the next-to-the-last well is mixed, take 60 μl of the 120 μl in this well and discard it. Leave the last well with 60 μl of DMSO/media diluent as a non-test compound-containing control. Make 9 columns of titrated test compound, enough for triplicate wells each for: (1) VEGF (obtained from Pepro Tech Inc., catalogue no. 100-200; (2) endothelial cell growth factor (ECGF) (also known as acidic fibroblast growth factor, or aFGF) (obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600); or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) and assay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-well assay plates containing the 0.8-1.0×104 cells/100 μl/well of the HUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO2.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, or media control to each test compound condition. As with the test compounds, the growth factor concentrations are 4× the desired final concentration. Use the assay media from day 0 step 2 to make the concentrations of growth factors. Incubate approximately 24 hours at 37° C., 5% CO2. Each well will have 50 μl test compound dilution, 50 μl growth factor or media, and 100 μl cells, which calculates to 200 μl/well total. Thus the 4× concentrations of test compound and growth factors become 1× once everything has been added to the wells.

The ability of human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xeno-transplantation of human tumors into athymic mice, (Rygaard and Poylsen, 1969, Acta Pathol. Microbial. Scand., 77:758-760), many different human tumor cell lines (e.g., mammary, lung, genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice. The following assays may be used to determine the level of activity, specificity and effect of the different compounds of the present invention. Three general types of assays are useful for evaluating compounds: cellular/catalytic, cellular/biological and in vivo. The object of the cellular/catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell. The object of the cellular/biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell. The object of the in vivo assays is to determine the effect of a compound in an animal model of a particular disorder such as cancer.

Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mM glutamine (GLN)). All cell culture media, glutamine, and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, N.Y.) unless otherwise specified. All cells are grown in a humid atmosphere of 90-95% air and 5-10% CO2 at 37° C. All cell lines are routinely subcultured twice a week and are negative for mycoplasma as determined by the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS or media (without FBS) to a particular concentration and the cells are implanted into the hindflank of the mice (8-10 mice per group, 2-10×106 cells/animal). Tumor growth is measured over 3 to 6 weeks using venier calipers. Tumor volumes are calculated as a product of length×width×height unless otherwise indicated. P values are calculated using the Students t-test. Test compounds in 50-100 μL excipient (DMSO, or VPD:D5W (U.S. Pat. No. 5,610,173 to Sxhwartz, et al.), can be delivered by IP injection at different concentrations generally starting at day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be used for the evaluation of therapeutic value and efficacy. of the compounds identified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimental animals. Implantation of tumor cells can be performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg Xylazine) are administered intraperitoneally. A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 107 tumor cells in a volume of 100 μl medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin is closed by using wound clips. Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurement of tumor size, grade of invasion, immunochemistry, in situ hybridization determination, etc.).

Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect. A measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index; i.e., IC50/LD50. IC50, the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein. LD50, the dosage which results in 50% toxicity, can also be measured by standard techniques as well (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol. Methods, 64:313; Decker and Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by measuring the lethal dose in animal models. Compounds with a large therapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50.

CONCLUSION

It will be appreciated that the compounds, methods and pharmaceutical compositions of the present invention are effective in modulating PK activity and therefore are expected to be effective as therapeutic agents against RTK, CTK-, and STK-related disorders.

One skilled in the art would also readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent herein. The molecular complexes and the methods, procedures, treatments, molecules, specific compounds described herein.are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional. features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described.

lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted lower alkoxy;

lower alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″, unsubstituted aryl or —NR13R14;

trihalomethyl;

unsubstituted alkenyl;

unsubstituted alkynyl;

unsubstituted aryl;

aryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl or lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ or NR13R14;

unsubstituted heteroalicyclic;

heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, —C(═O)H, —C(═O)—(unsubstituted lower alkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted aryloxy;

aryloxy substituted with a group independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxy and amino;

mercapto;

unsubstituted alkylthio;

unsubstituted arylthio;

arylthio substituted with one or more groups independently selected from the group consisting of halo, hydroxy and amino;

lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14;

unsubstituted lower alkoxy;

lower alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″, unsubstituted aryl or —NR13R14;

trihalomethyl;

unsubstituted alkenyl;

unsubstituted alkynyl;

unsubstituted aryl;

aryl substituted with one or more groups independently selected from the groups consisting of unsubstituted lower alkyl or lower alkyl substituted with a group selected from the group consisting of halo, —C(═O)OR″ or —NR13R14;

unsubstituted heteroalicyclic;

heteroalicyclic substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, —C(═O)H, —C(═O)—(unsubstituted lower alkyl), hydroxy, unsubstituted alkoxy, alkoxy substituted with a group selected from the group consisting of halo, —C(═O)OR″ and —NR13R14; unsubstituted aryloxy;

aryloxy substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halo, hydroxy and amino;

mercapto;

unsubstituted alkylthio;

unsubstituted arylthio;

arylthio substituted with one or more groups independently selected from the group consisting of halo, hydroxy and amino;

11. The compound or salt of claim 10 wherein at least one of R10, R11 or R12 is selected from the group consisting of —C(═O)OR″, —C(═O)O−M+, —(CH2)rC(═O)OR″ and —(CH2)rC(═O)O−M+.

12. The compound or salt of claim 11 wherein r of the —(CH2)rC(═O)OR″ or —(CH2)rC(═O)O−M+ group is 1.

13. The compound or salt of claim 11 wherein r of the —(CH2)rC(═O)OR″ or (CH2)rC(═O)O−M+ group is 2.

14. The compound or salt of claim 1, wherein said S-sulfonamido group is N,N-dimethylsulfonamido.

15. A pharmaceutical composition, comprising:

a therapeutically effective amount of the compound or salt of claim 1; and

a pharmaceutically acceptable carrier or excipient.

16. A method for the modulation of the catalytic activity of a protein kinase comprising contacting said protein kinase with a compound or salt of claim 1.

17. The method of claim 16 wherein said protein kinase is selected from the group consisting of a receptor tyrosine kinase, a non-receptor tyrosine kinase and a serine-threonine kinase.

18. A method for treating a protein kinase related disorder in an organism comprising administering a therapeutically effective amount of a compound or salt of claim 1 to said organism.

19. The method of claim 18 wherein said protein kinase related disorder is selected from the group consisting of a receptor tyrosine kinase related disorder, a non-receptor tyrosine kinase related disorder and a serine-threonine kinase related disorder.

20. The method of claim 18 wherein said protein kinase related disorder is selected from the group consisting of an EGFR related disorder, a PDGFR related disorder, an IGFR related disorder and a flk/KDR related disorder.

Design and synthesis of tetrahydropyridothieno [2, 3-d] pyrimidine scaffold based epidermal growth factor receptor (EGFR) kinase inhibitors: the role of side chain chirality and Michael acceptor group for maximal potency